We developed a method to study the existence and distribution of protein-free spaces in the cytoplasm, by the exclusion of probes of known dimensions from the chemically cross-linked and freeze-fractured cytoplasm matrix. The results were observed by electron microscopy and the probe utilized to explore the cytoplasm compaction was non-cationized ferritin (100 A degrees in diameter). We observed that cells in a resting state have crowded cytoplasm that after cross-linkage by glutaraldehyde is impermeable to ferritin. In contrast, ferritin freely penetrates the cross-linked cytoplasm of growing cells. Variations in cytoplasm permeability to ferritin were observed in single populations of lymphocytes from peripheral blood. These variations are in accordance with the heterogenous character of these populations. Results obtained with lymphocytes activated with Phytohaemagglutinin suggest that lymphocytes with permeable cytoplasms probably correspond to activated cells. Contrary to lumphocytes, homogenous populations of cells (neutophils from peripheral blood and cells from specific stages of differentiation of the fungus Phytophthora palmivora) respond uniformly to ferritin penetration. We also observed that cytoplasm compaction can change during differentiation and that the distribution of protein-free spaces in muscle cells characterizes different physiological states. We conclude the exclusion of probes from freeze-fractured cytoplasm is an easy and convenient method to evaluate cytoplasm compaction and to detect significant changes in the distribution of protein-free spaces at selected cellular states.